Project summary for: The role of the subpellicular microtubule array in Trypanosoma brucei cytokinesis Microtubules are essential cytoskeletal filaments in eukaryotic cells. They have an inherently dynamic nature and can rapidly alter their length to fulfill different functions in the cell. Often, microtubules are crosslinked into bundles to create stable structures for long-term force propagation, such as the mitotic spindle microtubules. The subpellicular microtubule corset of the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. The subpellicular microtubule corset maintains the highly asymmetric, tapered shape of T. brucei. The microtubules in the corset are crosslinked to each other and to the plasma membrane and do not break down at any point in the cell cycle; this puts unique constraints on cytokinesis in the parasite. However, it is not known how the array is replicated and segregated during cell division. Determining how the subpellicular array is built is of vital importance to understanding the life cycle of T. brucei, as its unique cell shape is required for its motility and pathogenicity. We have recently identified a microtubule-associated protein that localizes to the subpellicular array we called PAVE1. PAVE1 is necessary for the creation of the tapered shape of the trypanosome cell; when the protein is depleted, cells lack a posterior end, the subpellicular array becomes frayed, and cell division is inhibited. Thus, we hypothesize that PAVE1 is a microtubule crosslinking protein that promotes microtubule stabilization to form the tapered shape of the trypanosome posterior, which is essential for proper cytokinesis. We will conduct in vitro single-molecule assays with polymerized microtubules and heterologous expression of PAVE1 in mammalian cells to determine how PAVE1 affects microtubule behavior. Then, we will conduct live-cell experiments, pulse-chase assays, and serial block-face scanning electron microscopy to define how PAVE1 forms the subpellicular array, how the array is segregated during division, and why cell division is inhibited in cells lacking PAVE1. Elucidating PAVE1 function will reveal how trypanosomes build and maintain their remarkable subpellicular array, and inform on how microtubule crosslinking proteins change microtubule dynamics in general cell biology.